Ionic fluxes control both early and late stages of keratinocytes differentiation, directing process such as synthesis of differentiation-specific proteins and lipid secretion. Pharmacologic experiments, which demonstrated that an amiloride-sensitive ionic conductance controlled Ca2+ stimulated keratinocyte differentiation, led up to probe keratinocytes for the amiloride-blockable epithelial sodium channel (EnaC). EnaC alpha, beta and gamma subunits were identified in cultured keratinocytes and epidermis. Two aspects of EnaC subunit expression were dependent on the differentiation state of the keratinocytes. First, the beta subunit was expressed only in more differentiated keratinocytes, while the alpha and gamma subunits were expressed throughout differentiation. Second, alpha and beta subunits were expressed in adult but not fetal epidermis. Studies demonstrating hyperplastic, vacuolated epidermis in alpha-subunit knockout mice validated our hypothesis that this channel controls early aspects of differentiation. Further, electron micrography of this skin demonstrated an additional defect, i.e. premature lipid secretion. We will define EnaC channel properties and functions by comparing keratinocytes and epidermis of EnaC alpha-subunit knockout mice versus normal controls. We will examine how expression of different subunits during differentiation changes channel properties by comparing preconfluent versus post-confluent normal keratinocytes and by expressing these different subunit combinations in xenopus oocytes. Finally, we will define the keratinocyte EnaC channel regulation, testing three possible regulators: plasma membrane Ca2+ receptor, cAMP and stretch. This study will delineate the mechanism(s) by which this EnaC channel mediates keratinocytes differentiation.